The present invention relates to a method of modulating a release of biomolecules having heparin-binding affinity, and more specifically, to a method of modulating a release of biomolecules having heparin-binding affinity, using thiolated heparin adsorbed on metal surface.
A controlled release in response to external stimuli has been actively studied for the delivery of biomolecules such as proteins, DNA, peptides, and drugs. As external stimuli, pH change, temperature change, magnetic modulation, and electrical stimulation have been applied. Particularly, the advantage of electrical stimulation among external stimulations is that it can be applied locally within a designated region, with a precisely controlled time profile, thus spatiotemporal modulation is possible. In addition, electrical stimulation does not harm biological functions of biomolecules, cells, and organs in some limited ranges, so it is regarded as a relatively biocompatible means for in vivo application.
There have been many reports of electrochemically controlled release of charged molecules such as proteins and drugs based on polymers with two distinctive redox states, where one state is suitable for binding of target molecules and the other state accelerates the release of them. Electrodes serve to switch the redox states and the magnitude of applied current can control the release amount of molecules. For example, neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) incorporated into polypyrrole redox layer were released by the control of applied current. NT-3 and BDNF released from the polymer redox layer enhanced the survival of neuron and the elongation of neurites (Burgmayer P, Royce W. M. An ion gate membrane: electrochemical control of ion permeability through a membrane with an embedded electrode, Journal of the American Chemical Society 1982, 104, 6139-6140; Thompson. B. C, Richardson, R. T., Moulton, S. E., Evans, A. J, O'Leary, S, Clark, G. M., Wallace, G. G. Conducting polymers, dual neurotrophins and pulsed electrical stimulation-Dramatic effects on neurite outgrowth, Journal of Controlled Release 2010, 141, 161-167). Multilayer by layer-by-layer assembly was also employed to contain negatively charged Prussian blue nanoparticles as electroactive component and positively charged drugs via electrostatic interaction. Then, upon the application of electrical stimulation, multilayers were dissolved by a charge shift due to the oxidation of Prussian blue nanoparticles and the incorporated drugs were released accordingly (Daniel J. Schmidt, Joshua S. Moskowitz, Paula T. Hammond. Electrically Triggered Release of a Small Molecule Drug from a Polyelectrolyte Multilayer Coating, Chemical of Materials, 2010, 22, 6416-6425). These approaches have an advantage of incorporating a variety of molecules regardless of size, shape or chemical composition.
As an another approach of electrochemically controlled release of molecules, a reaction between gold and sulfur compounds has been used using the strong affinity of sulfur compounds to a transition metal surface. Examples of these surface active sulfur compounds include di-n-alkyl sulfide, thiophenols, mercaptopyridines, alkanethiolates, and cysteines. Among them, thiolates (RS—) have been studied extensively in recent years. They can be chemisorbed on gold surface by the oxidization of S—H group, which makes a quite strong binding (the absorption energy of Au—S bond is ≈44 kcal/mol.) (Dubois, L. H, Nuzzo, R. G. Synthesis, structure, and properties of model organic surfaces, Annual Review of Physical Chemistry, 1992, 43, 437-463). Especially, alkanethiols can easily form a uniform self-assembled monolayer (SAM) on gold surface, thus have been applied for molecular detections and recognitions, which can be detected by electrochemical measurement (Orozco, J, Medlin, L. K. Electrochemical performance of a DNA-based sensor device for detecting toxic algae, Sensors and Actuators B: Chemica 2011, 153, 71-77; Kim. G. I, Kim. K. W, Oh. M. K, Sung. Y M. Electrochemical detection of vascular endothelial growth factors (VEGFs) using VEGF antibody fragments modified Au NPs/ITO electrode, Biosensors and bioelectronics 2010, 25, 1717-1722; Miao. P, Liu. L, Nie. Y, Li. G. An electrochemical sensing strategy for ultrasensitive detection of glutathione by using two gold electrodes and two complementary oligonucleotides, Biosensors and bioelectronics, 2009, 24, 3347-3351).
More importantly, chemisorbed thiolates (RS—) can be electrochemically desorbed from gold surface by the reduction of sulfur when negative potentials are applied (Noshir S. Pesika, Kathleen J. Stebe, Peter C. Kinetics of Desorption of Alkanethiolates on Gold, Langmuir 2006, 22, 3474-3476; Ulman. A; Formation and Structure of Self-Assembled Monolayers, Chemical Review, 1996, 96, 1533-1554).RS—Au+e−RS−+Au
Thus, the electrochemical desorption of thiols has been applied as a means of controlled release of immobilized materials (e.g., biomolecules, channel compounds, DNAs, and subcellular release) by electrical stimulation. However, only burst release of immobilized molecules is possible by this method, and since alkanethiols are not perfectly biocompatible, the remaining alkanethiols along with the released molecules may cause problems in further use.
Heparin, a natural polysaccharide, has been used as an antithrombotic drug, but also has strong binding affinity for a variety of growth factors (e.g., endothelial growth factors (EGFs), fibroblast growth factors (FGFs), and hepatocyte growth factors (HGFs)) via specific heparin-binding domains in these molecules. Based on this specific binding with growth factors, heparin immobilization on various surfaces has been employed for controlled release of growth factors while maintaining their bioactivities (Hong S., Xixue H., Fei Y., Jianzhong B., Shenguo W. Cell affinity for bFGF immobilized heparin-containing poly(lactide-co-glycolide) scaffolds, Biomaterials, 2011, 32, 3404-3412; Cionne N. M, H. Mike K, Shelly S. E, Leesa M. G, Necat H, Stavros T. Sustained delivery of transforming growth factor beta three enhances tendon-to-bone healing in a rat model, wiley online library, 2011, 29,1099-1105). A method of releasing growth factors slowly using a heparin-based hydrogel was also disclosed as a conventional art, in which heparin is thiolated by modifying carboxylic acid group of heparin with cysteamine to prepare thiolated heparin, and then the thiolated heparin is cross-linked to form the heparin-based hydrogel (Tae, G, Kim, Y. J, Choi, W. I, Kim, M, Stayton, P. S, Hoffman, A. S. Formation of a novel heparin-based hydrogel in the presence of heparin-binding biomolecules, Biomacromolecules 2007, 8, 1979-1986). Here, thiolation of heparin reduces the anticoagulant activity of heparin, but the binding affinity with growth factors is not much affected by the thiolation. Whereas simple immobilization of heparin to surface of gold particles, etc. or the use of the heparin-based hydrogel can allow a temporary release or continuous sustained release of growth factors bound on heparin, these cannot modulate a release rate in accordance with time. Therefore, there are needs for methods for modulating the release rate of growth factors by an external signal, such as electrical stimulation, at a desired time.